Streptococcus pneumoniae is a major medical pathogen in the U.S., responsible each year for at least 500,000 hospitalizations and 40,000 deaths due to pneumonia, over 1 million U.S. clinic visits each year for the treatment of otitis media, and costs in excess of a billion dollars. Antibiotic resistance rates of pneumococcal isolates have soared to 20% or more in some areas of the United States. The goal of this project is to find and begin to characterize unknown genes that act as virulence factors in Streptococcus pneumoniae. Although several factors involved in virulence have been described, the pathogenesis of this organism is far from understood and additional virulence factors are likely to exist. The proposed experiments will identify genes that are expressed during animal infection but not in laboratory culture. Genes exhibiting an in vivo expression pattern are good candidates for new virulence factors. Examples might include genes encoding proteins involved in the transition from pneumonia to sepsis or from sepsis to meningitis. Genes specifically induced in the pneumococcal host will be identified by cloning their promoters upstream from a promoterless gene for resolvase, a site-specific recombinase that excises the DNA between direct repeats of a sequence called res. A S. pneumoniae reporter strain will carry a kanamycin resistance gene flanked by res sites in a genomic region unimportant for virulence. When the cloned promoters transformed into the reporter strain are active, resolvase will excise the kanamycin resistance gene through its action at the res sites. Thus, all descendent pneumococci will carry the evidence that the promoter had been active. Promoters induced in vivo will be isolated by selecting first against promoters that are active in culture broth with kanamycin, and then screening for kanamycin sensitive clones produced during infection in the mouse. Characterization of these promoters and the genes they control will lead to an increased understanding of the pathogenesis of S. pneumoniae.